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ORIGINAL RESEARCH published: 07 July 2021 doi: 10.3389/fgene.2021.643497

The Expression Profiles of mRNAs and lncRNAs in Buffalo Muscle Stem Cells Driving Myogenic Differentiation

Ruimen Zhang 1†, Jinling Wang 1†, Zhengzhong Xiao 2†, Chaoxia Zou 1, Qiang An 1, Hui Li 1, Xiaoqing Zhou 2, Zhuyue Wu 2, Deshun Shi 1, Yanfei Deng 1*, Sufang Yang 1,3* and Yingming Wei 1*

1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, China, 2 The Animal Husbandry Research Institute of Guangxi Autonomous, Nanning, China, 3 International Zhuang Medical Hospital Affiliated to Guangxi University Chinese Medicine, Nanning, China Edited by: Guohua Hua, Huazhong Agricultural Buffalo breeding has become an important branch of the industry. Hence, it University, China is of great significance to study buffalo production and meat quality. However, the Reviewed by: expression profiles of mRNA and long non-coding (lncRNA) molecules in muscle Ikhide G. Imumorin, stem cells (MuSCs) development in buffalo have not been explored fully. We, therefore, Georgia Institute of Technology, United States performed mRNA and lncRNA expression profiling analysis during the proliferation Bo Wang, and differentiation phases of MuSCs in buffalo. The results showed that there were China Agricultural University, China 4,820 differentially expressed as well as 12,227 mRNAs and 1,352 lncRNAs. *Correspondence: Yanfei Deng These genes were shown to be enriched in essential biological processes such as [email protected] cell cycle, p53 signaling pathway, RNA transport and calcium signaling pathway. We Sufang Yang also identified a number of functionally important genes, such as MCMC4, SERDINE1, [email protected] Yingming Wei ISLR, LOC102394806, and LOC102403551, and found that interference with MYLPF [email protected] expression significantly inhibited the differentiation of MuSCs. In conclusion, our research †These authors have contributed revealed the characteristics of mRNA and lncRNA expression during the differentiation of equally to this work buffalo MuSCs. This study can be used as an important reference for the study of RNA regulation during muscle development in buffalo. Specialty section: This article was submitted to Keywords: buffalo, muscle stem cells, mRNAs, non-coding RNAs, myogenesis Genomics, a section of the journal Frontiers in Genetics INTRODUCTION Received: 18 December 2020 Accepted: 25 May 2021 There is an annual increase in the global consumption of beef and it is an indispensable food in our Published: 07 July 2021 modern society, and therefore the beef cattle industry occupies an increasingly important position Citation: in modern agricultural practices (Bonny et al., 2015). According to statistics, in 2019, China’s beef Zhang R, Wang J, Xiao Z, Zou C, production was 6.85 million tons and beef imports were 1.66 million tons with a year-on-year An Q, Li H, Zhou X, Wu Z, Shi D, increase of approximately 57%. It is anticipated that China’s future beef demand will continue to Deng Y, Yang S and Wei Y (2021) The rise. Therefore, China urgently needs a viable and thriving beef cattle industry in order to provide its Expression Profiles of mRNAs and lncRNAs in Buffalo Muscle Stem Cells society with larger amounts of high-quality beef (Mwangi et al., 2019; Ornaghi et al., 2020). There is Driving Myogenic Differentiation. a need for us to conduct research on the growth and meat quality of locally produced beef as well as Front. Genet. 12:643497. to explore the potential molecular information of breeding stocks so as to provide reference values doi: 10.3389/fgene.2021.643497 for future breeding protocols (Grigoletto et al., 2020).

Frontiers in Genetics | www.frontiersin.org 1 July 2021 | Volume 12 | Article 643497 Zhang et al. Buffalo Differentiated MuSCs Expression Profiles

In ruminants, skeletal muscle tissue accounts for about 40– meat production. This culminated in the successful construction 60% of the adult animal body weight, which not only determines of the buffalo genomic DNA sequence map (Low et al., 2019). the level of meat production performance, but also has an Recently our laboratory analyzed the regulatory networks of important impact on meat quality. There is a group of myoblasts- lncRNA-mRNA interactions in the muscle tissue of cattle and muscle stem cells (MuSCs), which are the source of skeletal buffalo (Li et al., 2020). muscle formation and regeneration, and these have the potential However, when compared to cattle, buffalo muscle has the for differentiation and proliferation of muscle-derived stem cells characteristics of possessing greater shear force and consisting (Feige and Rudnicki, 2018; Feige et al., 2018). This is also the of thicker muscle fibers. At present, the molecular mechanisms current cell model for studying skeletal muscle development. that regulate buffalo muscle fibers formation are still unclear Under certain conditions, these cells can be activated causing the (Huang et al., 2021). We hypothesized that there are key MuSCs proliferate and differentiate. signaling pathway(s) which control the myogenic differentiation One of the main challenges in the field of muscle research is to of MuSCs. We, therefore, analyzed the mRNA expression understand how the genes that are involved in specialized muscle of MuSCs before and after myogenic differentiation through functions at the transcriptional and post-transcriptional levels transcriptome sequencing strategies in an attempt to screen the are regulated. Undoubtedly, myogenic regulatory factors (MRFs) signal pathways that may regulate muscle fiber development. (Hernandez-Hernandez et al., 2017), myocyte enhancer factor- Other recent studies have also shown that differential expression 2 (MEF2) (Taylor and Hughes, 2017), and PAX3/PAX7 genes lncRNAs also play an important physiological function during are the main genes involved in the growth and development of cellular differentiation of MuSCs (Zhu et al., 2017). This study skeletal muscle. Initially, long non-coding RNAs (lncRNAs) were further expands the understanding of skeletal muscle biology, considered to be transcriptional noise but later studies showed and provides a reference target for the genetic improvement of these RNAs play an important function in many biological buffalo and the production and cultivation of meat in vitro and processes (Jae and Dimmeler, 2020). Epigenetic control and in vivo. transcriptional regulation, , RNA metabolism, stem cell maintenance and differentiation, autophagy and apoptosis, embryonic development, and other aspects have also been shown MATERIALS AND METHODS to play important roles (Chen et al., 2020). With the discovery of a large number of important muscle regulators such as MuSCs Culture and Differentiation lncRNA H19 (Xu et al., 2017), Neat1 (Wang et al., 2019), lnc- All experiments regarding animals were performed in the State 133b (Jin et al., 2017), circLOM7 (Wei et al., 2017), more and Key Laboratory for Conservation and Utilization of Subtropical more ncRNAs related to muscle development have also been Agro-bio-resources, and were conducted in accordance with its widely characterized (Martone et al., 2019). At the same time, guidelines for the care and use of laboratory animals. Primary the important role of related coding RNAs, lncRNAs, and other water buffalo MuSCs were isolated and cultured from fetal- molecules in the development of skeletal muscle in agricultural derived longissimus muscle as described in Supplementary File animals are gradually being explored. 1, using a combination digestion method of type I collagenase So far, with the emergence of RNA structure detection and trypsin. MuSCs were cultured in high-glucose DMEM technologies such as Frag-seq (Underwood et al., 2010), supplemented with fetal bovine serum (Hyclone, USA; 10% FBS and 20% FBS, respectively) and antibiotics [1% penicillin and (ss/dsRNA)-seq, and SHAPE-seq, have allowed scientists to ◦ characterize the structure of RNAs obtained from different streptomycin; growth medium (GM)] at 5% CO2, 37 C. To tissues and cell components. When these data were combined induce MuSCs myogenic differentiation, MuSCs were switched with knowledge of RNA transformation events, such as miRNA to a differentiation medium (DMEM, 2% horse serum; DM) targeting, RNA modification, and the function of RNA binding when cells were almost 90% confluent for up to 4 days. (RPBs), they have emphasized the importance of RNA structure during regulation (Li et al., 2012). Moreover, most Sample Preparation of these studies are focused on mRNAs and ncRNAs in order to The tissues from Chinese buffalo at embryonic stage (90 days) explore the biological functions of RNA structure. were collected at a local in Nanning, Guangxi As a characteristic species of southern China, the potential use province. Tissue samples, including muscle, , heart, lung, of the buffalo as a meat source has gradually attracted attention. skin, kidney, brain, stomach, and intestine, were collected and The buffalo breeding industry has become a food basket project immediately frozen in liquid nitrogen. Proliferation of MuSCs for urban residents, but the meat production and meat quality of was labeled as the GM samples (n = 3) and differentiation of these buffalo needs to be improved for it to be an acceptable alternative was then called the DM samples (n = 3). The samples were kept to cattle (Li et al., 2020). Previously, several breakthroughs have at −80◦C until RNA was isolated. been made in studies of buffalo embryos, stem cells, and somatic cells, covering traits such as milk production, reproduction, and Total RNA Extraction Abbreviations: MuSCs, muscle stem cells; GO, ; KEGG, Kyoto Total RNA from cells and tissues samples were extracted with Encyclopedia of Genes and Genomes; lncRNAs, long non-coding RNAs; RT-qPCR, TRizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance quantitative real time PCR. with manufacturer’s instructions.

Frontiers in Genetics | www.frontiersin.org 2 July 2021 | Volume 12 | Article 643497 Zhang et al. Buffalo Differentiated MuSCs Expression Profiles

RNA-Seq and Transcriptome Data Analysis Femto reagent purchased from Thermo (Thermo Scientific, About 3 µg RNA per sample was used as the initial material Karlsruhe, Germany). for RNA sample preparation. PolyA-Seq libraries were prepared following the described protocol at RiboBio (Guangzhou, Vector Construction China) in accordance with the manufacturer’s instructions. Construction and sequencing identification of MYLPF The identification of mRNAs and lncRNAs was carried out interference vectors were completed by a Biological Company with reference to RiboBio’s technical methods. We have (GeneCopoeia, Guangzhou, China). The interference MYLPF provided a detailed description of the methods and analysis expression vector plasmids, which were named sh-MYLPF-A, sh- in Supplementary Table 9. All data were uploaded to the MYLPF-B, sh-MYLPF-C, and the control plasmids were named GEO database. NC. All primers sequences used are shown in Supplementary File 2. Analysis of Differentially Expressed Genes, Treatment of Cells mRNAs, and lncRNAs Muscle stem cells were grown to 70% confluence and then The RPKM (expected number of Reads Per Kilobase of transcript trypsinized and plated at 5 × 105 cells/well into six-well plates sequence per Millions base pairs sequenced) value was used to (Thermo Fisher Scientific, USA). They were then transfected with estimate the expression levels of mRNAs and lncRNAs. Genes vectors using X-treme GENE HP DNA Transfection Reagent with a RPKM value of <1 in no <50% of samples were defined (Roche, Basel, Switzerland). After incubation, the MuSCs were as unreliably expressed genes, while those with a RPKM value used for the different assays outlined below. In order to induce of ≥1 in more than 50% of samples were considered as reliably differentiation of myoblasts, the culture medium was changed to expressed genes. Differentially Expressed Genes DE mRNAs, and high-glucose DM medium. DE lncRNAs were analyzed using DESeq2, which defined them as reliably expressed genes with |log2 (Fold Change)|>1 and < Immunofluorescence and Microscopy Q-values 0.05 between any two groups. Myoblasts of MuSCs were washed three times with PBS buffer (pH 7.4), and permeabilized for 15 min in PBS containing Gene Ontology and KEGG Analysis 0.5% Triton X-100 before fixation in PBS containing 4% Gene ontology (GO; http://www.geneontology.org) and paraformaldehyde for 20–30 min. Immunostaining was carried KEGG pathway (http://www.kegg.jp) were analyzed as out as follows: cells were incubated overnight at 4◦C with described previously. primary anti-MyoD1 (1:200; Abcam) diluted in 5% bovine serum albumin. After that, cells were washed with PBS and Quantitative Real-Time PCR incubated at room temperature for 3–4 h with the corresponding Total RNA was extracted using TRizol reagent (Invitrogen, secondary antibody, goat anti-mouse IgG (H+L; 1:1,000; Carlsbad, CA, USA) according to the manufacturer’s Invitrogen) diluted in PBS. DNA was visualized using 5 instructions. Reverse was performed by using mg/ml DAPI staining. Finally, the prepared cells were washed the HiScript R II One Step RT-PCR kit (Vazyme, Nanjing, four times with PBS and observed under a fluorescence China). RT-qPCR was performed with ChamQ SYBR qPCR microscope (Nikon). Master Mix (Vazyme, Nanjing, China) using the 2− Ct method. Beta-actin was used as the internal control. All primer sequences Statistical Analysis used are listed in Supplementary File 2. The quantitative results are presented as means ± SEMs based on at least three independent experiments. Significant variance Western Blotting by treatments in comparison to the untreated samples was Cells were collected from different treatment groups, pelleted determined by one-way ANOVA performed with GraphPad by centrifugation, and then lysed in RIPA buffer. Total Prism version 8.0 (GraphPad Software, La Jolla, CA, USA). was prepared and protein concentrations were Differences were considered significant when P-values were determined using the Bradford method. Proteins were ≤ 0.05. then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to nitrocellulose RESULTS membranes. These were then blocked with 5% skimmed milk powder solution for 1.5–2 h at room temperature. The Variation of Phenotypic Characteristics membranes were then incubated overnight with primary During Differentiation of Buffalo MuSCs antibodies. Anti-PCNA, anti-CDK2, and anti-β-actin were A combination digestion method of type I collagenase and purchased from Abcam (Cambridge, MA, USA). After that, trypsin was used to obtain buffalo fetal-derived MuSCs. the membranes were washed with PBS-tween and incubated This cell type is similar to fibroblasts and spindle-shaped for 1.5 h with horseradish peroxidase-conjugated secondary in appearance. These cells have good proliferation capacity antibodies (Abcam, Cambridge, MA, USA). Protein bands (Figure 1A), which is referred to as the proliferation phase were detected after treatment with Super Signal West (GM samples) of MuSCs. In addition, when the medium was

Frontiers in Genetics | www.frontiersin.org 3 July 2021 | Volume 12 | Article 643497 Zhang et al. Buffalo Differentiated MuSCs Expression Profiles

FIGURE 1 | Analysis of the characteristics of buffalo MuSCs during proliferation and differentiation. (A) Proliferation phenotype of muscle stem cells (GM samples). (B) Differentiation phenotype of muscle stem cells (DM samples). (C) MuSCs grown as GM and DM samples were subjected to real-time PCR for BCL-2 and Pax7. (D) The differentiation phenotype (DM) samples of MuSCs were subjected to real-time PCR for MyoD1, MyoG, and MyHC. (E,F) MuSCs grown as GM and DM samples were subjected to western blotting for determination of PCNA and CDK2 proteins. (G) The differentiation phenotype (DM) samples of MuSCs were subjected to immunofluorescence for MyOD1. The data are presented as means ± SDs, n = 3 per group. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars = 100/200 µm. replaced with DM, after 2 days, the cells began to show that the cells obtained were MuSCs with the capability of myotube fusion. On the fifth day, the number of myotubes myogenic differentiation. increased and the myotube fusion became more obvious, which is referred to as the differentiation phase (DM samples) of MuSCs (Figure 1B). PolyA-Seq Characteristics of Buffalo Western blotting showed that the expression of PCNA MuSCs and CDK2 in MuSCs GM samples were significantly higher In order to identify the mRNAs and lncRNAs involved than that in DM samples (Figures 1E,F). The expression in proliferation and differentiation, we compared the levels of BCL-2 and Pax7 (paired Homeobox transcription polyA-seq status of GM and DM samples of MuSCs factors) in GM samples were significantly higher than those (Supplementary Figure S1). Analysis of sequencing data in MuSCs DM samples (Figure 1C). Immunofluorescence revealed that a very large number of clean reads, total maps, experiments showed that the muscle marker molecule, MyOD1, and uniquely mapped areas were involved in these processes was enriched in DM samples of MuSCs (Figure 1G). The (Figure 2A). The analysis of uniquely map profiles of MuSCs, expression levels of muscle-derived marker molecules, showed the distribution for the reads in different MYOD1, MYOG, and MyHC, increased significantly in (Figures 2B,C). Among them, most of the reads from GM and DM samples of MuSCs (Figure 1D). These results suggest DM samples were found to be targeted to exonic areas.

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FIGURE 2 | RNA-seq analysis of buffalo MuSCs during proliferation and differentiation. (A) RNA-seq information statistics of samples. (B) Mapped reads distribution. (C) The distribution for the reads in different chromosomes.

TABLE 1 | PolyA-seq statistics of the different results obtained. correlation analysis on these lncRNAs. We performed GO and KEGG enrichment analysis on the related regulatory DEGs in Differential type Upregulated Downregulated Total the processes of proliferation, differentiation, transformation, Genes 2,979 1,841 4,820 and maturation. This produced signal pathway information mRNAs 7,505 4,722 12,227 which was then used to predict the functions and mechanisms lncRNAs 831 521 1,352 of the mRNAs (Supplementary Tables 5, 6). The results of pathway analysis of DEGs showed that GO analyzed and annotated these into three main categories: biological processes, Profiles of DE Genes, mRNAs, and lncRNAs cellular components, and molecular functions, including ATP During Differentiation of Buffalo MuSCs binding and nucleus RNA-directed DNA polymerase activity Analysis of sequencing data revealed that a total of (Figure 3). In addition, we employed KEGG pathway enrichment 31,819 genes, 57,640 mRNAs, and 11,357 lncRNAs were analysis to further understand the biological functions and involved in the proliferation and differentiation of MuSCs molecular interactions of most DEGs with the assumption that (Supplementary Table 1). We also performed heatmaps the identified pathways may be involved in the development and volcano plots for the genes, mRNAs, and lncRNAs and growth of buffalo skeletal muscle. We found more than in MuSCs (|log2 (FoldChange)|>1, Q-value <0.05) 300 pathways to be enriched, and the top 30 most significant (Supplementary Figure S2). There were 4,820 DEGs, 12,227 terms were uncovered, including biological processes such DE mRNAs, and 1,352 DE lncRNAs (Supplementary Tables 2– as cell cycle, p53 signaling pathway, RNA transport, and 4). Compared with the GM samples of MuSCs, 2,979 genes calcium signaling pathway (Figure 4). In short, these signal (61.80%), 7,505 mRNAs (61.38%), and 831 lncRNAs (61.46%) pathways related to DEGs play an important role in the were upregulated, while 1,841 genes (38.20%), 4,722 mRNAs regulation of MuSC proliferation and differentiation, which (38.62%), and 521 lncRNAs (38.54%) were downregulated in provides an important basis for subsequent research on DM samples of MuSCs (Table 1). buffalo myogenesis.

Signal Pathway Enrichment Analysis of The Verification of DEGs and DE lncRNAs DEGs Between Proliferation and in MuSCs Differentiation Phases of Buffalo MuSCs Based on the expression levels of DEGs and DE lncRNAs, Since we mainly analyzed mRNAs transcripts, and also involved a including 12 genes (MCM4, MCM7, SERDINE1, SEMA7A, small number of known lncRNAs, we did not perform functional C1QTNF6, CPZ, VDR, PLAC9, ISLR, MyOG, PCNA, and

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FIGURE 3 | Gene ontology (GO) analysis. (A) GO analysis of DEGs in the GM samples of MuSCs. (B) GO analysis of DEGs in the DM samples of MuSCs.

FIGURE 4 | Kyoto Encyclopedia of Genes and Genomes pathway analysis. (A) Top 30 KEGG terms of DEGs in the GM samples of MuSCs. (B) Top 30 KEGG terms of DEGs in the DM samples of MuSCs.

cyclin D1) related to the cell cycle, actin cytoskeleton, cell The Role of MYLPF in Buffalo MuSCs differentiation, and lipid metabolism (Figures 5A,B), and We identified a dysregulated gene, MYLPF, which was shown to seven random lncRNAs (LOC102403551, LOC112586870, be upregulated significantly (by almost 60-fold) in DM compared LOC112584513, LOC102399397, LOC112581569, with GM samples when measured by RT-qPCR (Figure 6A). LOC102395296, and LOC102394806; Figure 5C), were In addition, MYLPF was expressed in various tissues, such as selected for RT-qPCR verification. After comparisons with heart and liver and the highest expression levels seen in muscle the RNA-seq data, similar expression trends for RT-qPCR were (Figure 6B). A previous report also showed that the relationship discovered, showing the strong consistency between RT-qPCR between MYLPF and meat quality can be used as an important and RNA-seq data. genetic consideration when dealing with gene-assisted selection

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FIGURE 5 | Validation of the expression levels of DEGs, DE lncRNAs by RT-qPCR. (A,B) The validation results of DEGs and (C) DE lncRNAs. The data are present as means ± SDs, n = 3 per group. ***P < 0.001.

Frontiers in Genetics | www.frontiersin.org 7 July 2021 | Volume 12 | Article 643497 Zhang et al. Buffalo Differentiated MuSCs Expression Profiles programs. This suggests that MYLPF may play an important role and they affected the formation of myoblasts and the fusion of in muscle development. Therefore, MYLPF was selected for more myotubes. In addition, we performed target gene analysis on in-depth study in order to further explore its potential functions differentially expressed lncRNAs, and obtained many lncRNAs- in MuSCs. target gene relationship networks. We can indirectly predict Subsequently, interference vector (sh-MYLPF-A/B/C/NC) the function of the candidate lncRNA through the target gene plasmids were introduced into the 293T cell line which is derived (Supplementary Tables 7, 8). Previous studies had confirmed from embryonic kidney 293 cells and contains the SV40 that compared with cattle, buffalo muscles have larger muscle T-antigen. After 24 h, the reporter gene, green fluorescence fiber diameters and poorer meat texture. Of course, myotube protein (GFP), was found to be expressed in the transfected fusion is an important factor affecting the formation of muscle cells, with a strong fluorescence observed under the fluorescence fibers (Picard and Gagaoua, 2020). The mRNAs and lncRNAs microscope (Supplementary Figure S3). Further qPCR showed which are related to myogenic differentiation of MuSCs may that the expression levels of MYLPF declined by 70% in the regulate the diameter of muscle fibers through myotube fusion, transfected cells with the sh-MYLPF-A plasmid (Figure 6C). which further affected the quality of meat. We also found some Sh-MYLPF-A vectors were then transferred into P2 MuSCs key signal transduction pathways, such as p53 signal transduction (Supplementary Figure S4). The cells were cultured for a further pathway, TGF-β signal pathway, calcium signal transduction 24 h, followed by replacement of the medium with myogenic pathway that were related to these RNAs. These signal pathways differentiation medium. On the fourth day, the knockdown of are involved in cell development and maintenance of muscle MYLPF was found to have inhibited the formation of myotubes structure and function, suggesting that they were also likely to be (Figure 6D). The marker gene of myoblast differentiation, important regulatory signals for regulating buffalo muscle fiber MyoD1, was then measured by qPCR. The results showed that hypertrophy (Liu et al., 2018; Valle-Tenney et al., 2020). there were significantly lower levels of MYLPF in the knockdown We also randomly selected a batch of candidate molecules group compared to the controls (Figure 6E). These findings for verification, and their expression trends were found to suggested that MYLPF knockdown inhibited differentiation be consistent with the RNA-seq results, indicating that the of MuSCs. sequencing data was reliable. We found that genes such as VDR, PLAC9, ISLR were involved in the myogenic differentiation process of MuSCs, but their molecular mechanisms needed DISCUSSION to be further explored ( et al., 2020; Cui et al., 2020). It had been confirmed that the immunoglobulin superfamily Currently, the global population is 7.7 billion, and it is expected containing -rich repeats (ISLR) promoted skeletal muscle to exceed 9 billion by 2050 (Bonny et al., 2015). By then, mankind regeneration by activating canonical Wnt signaling. Loss of will face a bigger challenge of food provision for the growing function of ISLR resulted in defective differentiation of myoblasts population, and this will have a major impact on global meat leading to a block in myotube formation (Zhang et al., 2018). consumption which will increase accordingly (Ornaghi et al., Therefore, ISLR may be an important biological regulator to 2020). Muscle development is an important factor that affects control buffalo muscle development. It had also been reported the growth rate, meat yield, meat quality, and other important that MYLPF was one of the muscle-derived marker genes economic traits of livestock, and this process is dependent on the involved in the process of muscle metabolism and related to meat proliferation and differentiation of MuSCs (Feige and Rudnicki, quality traits (Rosa et al., 2018; Chong et al., 2020). As one of 2018; Boscolo Sesillo et al., 2020). the muscle markers, MYLPF is expected to become a target for Initially, we established a successful protocol for in vitro regulating the quality traits of buffalo meat (Silva et al., 2019). culture of buffalo MuSCs, which provided a good working We also found that decreased MYLPF was linked to an inhibition foundation for subsequent research on candidate factors that of myogenic differentiation of buffalo MuSCs, but the molecular regulate buffalo muscle development. With the rise of in vitro mechanism of this phenomenon is not yet fully understood. such as laboratory meat, the role played by Therefore, how MYLPF regulates buffalo muscle regeneration is MuSCs is becoming more important. The in vitro cultured meat worthy of further investigation. production technology is still in its infancy, and it is necessary At present, lncRNA is also one of the research hotspots in to strengthen and improve the technical systems involved in the field of ncRNA (Martone et al., 2019). However, we only MuSCs production of beef (Bhat et al., 2017). Therefore, buffalo discovered the number of known lncRNAs and their expression MuSCs can play an important role in the emerging research levels involved in the myogenic differentiation of MuSCs. fields of animal husbandry, such as providing improvement of Then, we screened out a batch of potential candidate lncRNAs, buffalo meat quality and production as well as increasing our such as LOC102403551, LOC112586870, and LOC102394806. biochemical knowledge of MuSCs in vitro. These potential candidate lncRNAs may affect the myoblast In this study, we constructed the expression profiles of mRNAs differentiation of MuSCs by regulating through and lncRNAs in the process of myogenic differentiation of miRNAs, RPBs, and other ways (Chi et al., 2019; Xu et al., 2019; buffalo MuSCs. During this process, a total of 4,820 genes, Guo et al., 2020; Liu et al., 2020). In future studies, we and 12,227 mRNAs, and 1,352 lncRNAs were differentially expressed. others will also investigate the interactions between lncRNAs and Among them, 2,979 genes, 7,505 mRNAs, and 831 lncRNAs were enhancers in order to regulate fate of MuSCs (Lin et al., 2019; significantly related to the myogenic differentiation of these cells, Nikonova et al., 2019; Williams et al., 2020). The biological effects

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FIGURE 6 | Expression and characterization of MYLPF in buffalo skeletal muscle. (A) Validation of the expression of MYLPF by RT-qPCR. (B) The expression levels of MYLPF in different tissues of buffalo. (C) The interference efficiency of MYLPF was measured by RT-qPCR. (D) Inhibition of MYLPF expression on myotubule formation. (E) The mRNA expression of myogenesis marker gene, MyOD1, was measured by RT-qPCR. The data are presented as means ± SDs, n = 3 per group. *P < 0.05; **P < 0.01. Scale bars = 100 µm. of these lncRNAs related to buffalo MuSCs, lncRNA evolution, buffalo with a view to improving its share as a desirable beef lncRNA SNP issues, etc. are also worth pursuing (Qian et al., alternative in the marketplace. 2019). At the same time, how these lncRNAs and coding genes regulate the molecular mechanisms of farmed beef production DATA AVAILABILITY STATEMENT and their contributions to the in vitro meat production process also need to be further explored. The datasets presented in this study can be found in online In summary, we have established the mRNA and lncRNA repositories. The names of the repository/repositories and expression profiles that regulate the myogenic differentiation accession number(s) can be found below: NCBI GEO, accession of buffalo MuSCs, and further predicted and verified the no: GSE164808. signaling pathways and candidate regulators involved in cell proliferation and differentiation. These results enrich the AUTHOR CONTRIBUTIONS expression information of factors that regulate the development of MuSCs in Chinese local fine beef cattle breeds, and provide YD, SY, YW, JW, and RZ conceived and designed the effective genetic information for future programs of breeding experiments. RZ, JW, and CZ performed the experiments and high-yield beef cattle. analyzed the data. RZ, QA, CZ, XZ, ZW, HL, and DS contributed reagents, materials, and helped to analyze the data. RZ and CONCLUSIONS ZX wrote the manuscript and SY, YD, DS, and YW revised it. All authors contributed to the article and approved the In this study, the proliferation and myogenic differentiation submitted version. phenotypic characteristics of buffalo MuSCs were compared for the first time, and the expression of mRNAs and lncRNAs in FUNDING these cells were reviewed. Many coding RNAs and lncRNAs were found to be differentially expressed during the proliferation and The research was funded by the National Natural myogenic differentiation phases of MuSCs. We further identified Science Foundation of China (31860644 and 31760334), and verified a number of differentially expressed molecules such Natural Science Foundation of Guangxi Province as SERDINE1, ISLR, MYLPF, LOC102403551, LOC112586870, (AA17204051, AA17204052, 2017GXNSFBA198170, and and LOC112584513. This study lays the foundation for further 2019GXNSFDA185002), Nanning Scientific Research research on the role of lncRNAs in the muscle development of Technological Development Foundation (20194147 and

Frontiers in Genetics | www.frontiersin.org 9 July 2021 | Volume 12 | Article 643497 Zhang et al. Buffalo Differentiated MuSCs Expression Profiles

20192087), and Guangxi Innovation Team Construction Project grateful to Dr. Dev Sooranna of Imperial College London for of National Modern Agricultural Industry Technology System English language edits of the manuscript. (nycytxgxcxtd-09-01). SUPPLEMENTARY MATERIAL ACKNOWLEDGMENTS The Supplementary Material for this article can be found We would like to thank RiboBio (Guangzhou, China) for online at: https://www.frontiersin.org/articles/10.3389/fgene. conducting the whole-genome RNA sequencing. We are also 2021.643497/full#supplementary-material

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Frontiers in Genetics | www.frontiersin.org 10 July 2021 | Volume 12 | Article 643497 Zhang et al. Buffalo Differentiated MuSCs Expression Profiles

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Frontiers in Genetics | www.frontiersin.org 11 July 2021 | Volume 12 | Article 643497